The long-term goal of this project is to develop a detailed molecular understanding of the CLC (Chloride Channel) family of membrane proteins. The CLCs comprise two major classes of ion-transport mechanisms: half of CLC homologs are electrodiffusive ion channels (catalyzing downhill movement of chloride), while the other half are secondary active transporters that stoichiometrically exchange chloride for protons (harnessing the energy from movement of chloride to pump protons or vice versa). That both types of ion-transport are within one gene family suggests their mechanisms may be subtle variations on a single central theme. Indeed, CLC channels appear to act by a broken transporter mechanism. Here we propose a highly concerted approach composed of complementary computational and experimental biophysical and biochemical techniques to study the molecular details underpinning the mechanism of CLC-ec1 and CLC-0, model homologs for antiporters and channels, respectively. Our main goal is to elucidate the antiporter (unbroken) mechanism, taking advantage of high-resolution CLC-ec1 structures and the molecular dynamics simulations they allow, and of antiporter amenability to spectroscopic analysis. We will apply insights from studies of the unbroken transporter CLC-ec1 to electrophysiological analysis of the CLC-0 channel's broken mechanism to study conservation between channel and transporter mechanisms. AIM 1 will determine global structural changes associated with the CLC transport cycle. Here we will use EPR to measure distance changes between pairs of site-directed spin labels on CLC-ec1, evaluate changes in accessibility of spin labels, and use computational modeling to develop structural models for the inward- and outward-facing states. AIM 2 will determine how CLC conformational change affects water dynamics and water-wire formation involved in proton transport. These studies will help reveal how proton transport fits into the overall CLC transport mechanism. AIM 3 will characterize the chloride/proton coupling mechanism ? evaluating detailed models of how transport occurs, using a combination of kinetic and spectroscopic measurements on WT and uncoupled mutants, together with computational analysis to investigate in detail how binding and translocation of ions are coupled to protein conformational changes. Overall Impact: Revealing molecular details of CLC ion channel broken and antiporter unbroken mechanisms, and how they are alike and different, will help reveal how CLC function can go wrong, with implications for neurological diseases, hypertension, and diseases of kidney, muscle, and bone. Our methodology will be applicable to other large membrane proteins of medical importance where unraveling molecular mechanisms has similarly been stymied by limitations of crystallography.